The mechanical stiffness of DNA in?uences gene expression through effects on nucleosome positioning and DNA looping. However, four major gaps remain in our understanding of DNA stiffness. We do not understand what chemical features of DNA control its stiffness. We do not understand if simple polymer models are adequate to describe biologically-relevant DNA looping. We do not understand why DNA can appear softer in vivo than in vitro. Finally, we do not understand the detailed mechanisms of DNA softening by sequence-nonspeci?c architectural proteins. Insight into these four problems promises multiple impacts. For example, DNA-like polymers might be designed with programmed ?exibility, DNA looping might be controlled by sequence-targeted DNA bending proteins, and tighter gene control switches might be engineered. Our laboratories have a proven track record of collaborative experiments to probe the origin of DNA mechanical properties, and how these properties are modi?ed by architectural proteins in cells. During the previous funding period we made important progress and now propose four aims to continue this fundamental research. Aim 1 will map architectural protein binding on tightly-looped DNA in living E. coli cells. Aim 2 will develop a novel massively parallel approach to measure DNA looping energetics and test predictions of the wormlike chain polymer model. Aim 3 will map the topology of repression loops in the DNA of living bacteria to test a hypothetical explanation for why DNA appears softer in vivo than in vitro. Finally, Aim 4 will apply single molecule and biochemical approaches to explore how sequence nonspeci?c high mobility group (HMGB) proteins modify the mechanical properties of DNA and chromatin in eukaryotes.